Flow Cytometry in Drug Discovery and Development

Virginia Litwin, Philip Marder, Virginia Litwin, Philip Marder

• Publisher: Wiley
• Year: 2011
• Print ISBN: 9780470433560
• eBook ISBN: 9780470922781
• Edition: 1
• Topic: Medicine
• Subtopic: Pharmacology

This book covers the unique application of flow cytometry in drug discovery and development. The first section includes two introductory chapters, one on flow cytometry and one on biomarkers, as well as a chapter on recent advances in flow cytometry. The second section focuses on the unique challenges and added benefits associated with the use of flow cytometry in the drug development process. The third section contains a single chapter presenting an in depth discussion of validation considerations and regulatory compliance issues associated with drug development.

Part I

Introduction

Philip Marder and Virginia Litwin

The discovery and development of novel therapeutic compounds is a lengthy, difficult, and expensive process with recent estimates of more than 1.2 billion dollars required for each new drug brought to market. As a result, the standard processes of the pharmaceutical industry are being reevaluated and modified in order to increase efficiencies in the drug development process. One approach in process transformation is to promote more informed decision making by incorporating advanced technologies such as flow cytometry.

A wide variety of flow cytometric methods are employed during various stages of the drug development life cycle. This book explores many of the benefits and complexities associated with this unique application of the technology. Part I is intended to provide the reader with essential background information regarding both flow cytometry (Chapters 1 and 2) and drug development (Chapter 3).

Chapter 1

Introduction to Flow Cytometry

Elizabeth Raveche, Fatima Abbasi, Yao Yuan, Erica Salerno, Siddha Kasar, and Gerald E. Marti

1.1 Introduction

This chapter presents, in basic terms, the concepts and principles of flow cytometry. Numerous books and articles describing flow cytometers and their use in a clinical and biomedical research setting have been published [1–7]. In this chapter, flow cytometers will be discussed from their infancy arriving at the current instrumentation that allows for detection of numerous features of individual cells or particles, including determination of size and granularity, surface marker expression, DNA content, intracellular protein expression, and function. The key to flow cytometers is that the analysis is done on cells in suspension [8–10]. The analysis of individual cells (or particles) rather than the whole population allows for detection of multiple properties measured on the same cell. The detection is rapid (as fast as the cell in the fluid sheath passes through the laser beam). In addition to analysis of individual cells, some types of flow cytometers can physically sort cells based on signals associated with the parameters being detected. The term fluorescence-activated cell sorter or FACS has been adopted to refer to this type of analysis [11]. Flow cytometry is a very useful tool for both clinical diagnosis and scientific research. The history of flow cytometers has been the subject of numerous reviews [12–20]. The first flow cytometers were introduced in the mid-1970s and first used for DNA analysis and leukemia immunophenotyping [7, 21–25]. A further impetus to bring flow cytometers to the forefront of clinical labs came in the early 1980s with the discovery that individuals infected with the HIV virus developed AIDS, which could be monitored by enumerating the number of CD4⁺ T cells by flow cytometric analysis [26–30]. Currently, there are emerging areas with flow cytometric applications including the enumeration of CD34⁺ hematopoietic stem cells [29, 31, 32], detection of circulating metastatic tumor cells [33–37], determination of antigen-specific T cells [38–40], and identification of pathogens [41–45], to list a few. Combination of sorting with molecular analysis represents an important use of the sorting aspects of flow cytometers. There are over 100,000 flow cytometers in use and the employment of this instrument in clinical diagnostics has increased dramatically, particularly with the increase in FDA-approved fluorochrome reagents for in vitro diagnostics (fluorochrome-conjugated antibodies). However, in third-world countries, access to clinical flow cytometers is not optimal [46, 47]. The use of flow cytometers and the impact of this instrument on biomedical and clinical studies can be appreciated by looking at the increase in publications in which the word “flow cytometry” appeared in the abstract or title with time (Figure 1.1).

Figure 1.1 A bar graph showing the number of publications having “flow cytometry” in their title/abstract since 1970 to present. There is almost a 150% increase since 1980–1989.

Improvements in instrumentation and computer-assisted analysis have made the flow cytometer a critical instrument in biomedical research, clinical diagnostics, and drug discovery. Herzenberg was honored for his work in flow cytometry by the American Association for Clinical Chemistry with the Ullman Award in 2002 and some of the history described in this chapter comes from his lecture and the accompanying article [19]. The original description of the first flow cytometer was provided in Scientific American [48]. This instrument consisted of one laser and two light detectors, one for forward scatter to measure cell size and the other for fluorescence. This meant that one was restricted to measuring a single marker. When one of the authors of this article used that prototype instrument, the LASL, we were measuring the DNA content of individual cells. This was one of the first uses of these early flow cytometers since reagents were available that not only bound specifically to DNA (e.g., ethidium bromide developed by Dittrich and Gohde in 1969 [49]) but also emitted fluorescence when excited with a laser. Much of the essentials of the modern-day FACS are the same as those in the early flow cytometers. However, these early flow cytometers were cumbersome and required an on-site engineer. The laser was water cooled and alignment issues were critical. In addition, no computer was attached to these early flow cytometers, nor were programs available for data analysis [50]. At one point, we took Polaroid pictures of oscilloscopes and sent data to a DEC10 supercomputer and wrote our own programs for cell cycle analysis.

Although the development of FACS depended on many advances in various disciplines including dye chemistry, electronics, and computers, one important breakthrough that was critical for the development of flow cytometers was the principle of measuring cells or particles in liquid suspension. Advances in the flow principle began in 1940 with Crosland-Taylor using the flow principle and light scatter to measure blood cells [51]. The breakthrough technology was first developed by Coulter and the Coulter principle describes changes in the electrical conductivity of a small saline-filled orifice as a cell passes through it. In 1953, Wallace Coulter and his brother Joe obtained a U.S. patent for the Coulter counter that automated counting of particles, particularly cells in the blood [52]. The use of a liquid stream (or a sheath) to which a sample is introduced allows individual cells to be distributed in the sheath that then passes through a nozzle (detecting electrical conductivity changes) to generate a trigger, which indicates the presence of a signal that exceeds the threshold level.

Many of the applications for FACS analysis involve the identification of membrane markers via the use of fluorochrome-tagged antibodies, which recognize these markers. Many of these membrane markers are surface proteins or surface antigens, which help to define the cell. These antigens are used to classify the cells and are often assigned a cluster of differentiation number or a CD number. Antibodies (which are normally produced by B lymphocytes) can be made that specifically bind to these CD molecules. There are more than 200 CD molecules that have been identified and specific antibodies have been produced that recognized these CD markers [53–55]. In addition, many of these antibodies are commercially available as labeled antibodies with different fluorochromes.

1.2 Basic Principles of How a Flow Cytometer Works

The basic components of a flow cytometer (Figure 1.2) consist of (1) a flow cell that forces single cells into the middle of a fluidic sheath, (2) a laser source of light, (3) optical components to focus light of different wavelengths (colors) onto a detector, (4) a photomultiplier to amplify the signal, and (5) a computer.

Figure 1.2 Diagrammatic representation of a basic flow cytometer. The fluorescently labeled cells are hydrodynamically focused into a single file in the flow cell. Individual cells are excited by the laser light source and the fluorescence emissions, FSC, and SSC are detected. The cells can then be given a particular charge based on their fluorescence profile and deflected toward the oppositely charged plates. In the figure, light grey cells and dark grey cells are given negative and positive charges, respectively, and are thus deflected toward two different tubes.

In a basic flow cytometer, the sample (containing the cells tagged with fluorochromes in a liquid) is drawn up and pumped into the flow cell through tubing. The cells flow through the flow chamber rapidly and singly and are passed through one or more laser light beams. As the laser beam hits the cells, the light beam is scattered in a forward direction and a side direction. Fluorescence emission can also be detected. Scatter or fluorescence is captured, filtered (based on the wavelength), and directed to the appropriate photodetectors for conversion to electronic signals. The electronics in the flow cytometer amplify the signal and convert the analog data to digital data, which can then be analyzed by computer software programs.

1.3 Fluidics

1.3.1 Flow Cells

In order to perform flow cytometric analysis, the sample must be in a suspension and the cell in the sample stream must be centered in the laminar flow [49]. Hydrodynamic focusing induces cells to orient with their long axis parallel to the flow. The end result is that the introduced sample passes by the laser with each cell oriented in the center of the sample stream in a particular manner in three dimensions.

1.4 Optics

Flow cytometers depend on the laws of optics, such as reflection, refraction, and other principles, which are not new but based on works established centuries ago [56]. Optics are present on both the excitation and the emission side. The excitation optics encompass the lasers and the lenses that focus the laser beam. The emission optics are involved in collecting the emission following excitation. These involve lenses to collect emitted light and mirrors and filters to route specified wavelengths of the collected light to designated optical detectors. Light coming out of a laser may be considered a beam but fluorescence must be considered as a photon.

1.4.1 Light Scatter

Due to differences between the refractive indices of cells and the surrounding sheath fluid, light impinging upon the cells is scattered. The forward light scatter (FSC) provides empirical information on cell size. Light scattered in an orthogonal direction or side scatter (SSC), which is collected by a different detector, provides information about granularity.

1.4.2 Types of Lasers

Laser stands for light amplification by stimulated emission of radiation. Gas lasers have mirrors at each end of a cylinder or plasma tube filled with an inert gas. The gas is ionized to a higher energy state by a high-voltage electric current. When these excited atoms return to the ground state, they give off photons of a charact...

Table of contents

• Cover
• Title Page
• Copyright
• Dedication
• Preface
• Foreword
• Acknowledgments
• Contributors
• Part I: Introduction
• Part II: Flow Cytometry in the Drug Development Process
• Part III: Validation and Regulatory Compliance
• Part IV: Future Directions
• Color Plates
• Index